Internalization and Subcellular Trafficking of Poly - American Chemical

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Internalization and Subcellular Trafficking of Poly‑L‑lysine Dendrimers Are Impacted by the Site of Fluorophore Conjugation Brittany R. Avaritt and Peter W. Swaan* Department of Pharmaceutical Sciences, Center for Nanobiotechnology, University of Maryland, Baltimore, 20 Penn Street, Baltimore, Maryland 21201, United States ABSTRACT: Internalization and intracellular trafficking of dendrimer−drug conjugates play an important role in achieving successful drug delivery. In this study, we aimed to elucidate the endocytosis mechanisms and subcellular localization of poly-L-lysine (PLL) dendrimers in Caco-2 cells. We also investigated the impact of fluorophore conjugation on cytotoxicity, uptake, and transepithelial transport. Oregon green 514 (OG) was conjugated to PLL G3 at either the dendrimer periphery or the core. Chemical inhibitors of clathrin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis pathways and macropinocytosis were employed to establish internalization mechanisms, while colocalization with subcellular markers was used to determine dendrimer trafficking. Cell viability, internalization, and uptake were all influenced by the site of fluorophore conjugation. Uptake was found to be highly dependent on cholesterol- and dynamin-mediated endocytosis as well as macropinocytosis. Dendrimers were trafficked to endosomes and lysosomes, and subcellular localization was impacted by the fluorophore conjugation site. The results of this study indicate that PLL dendrimers exploit multiple pathways for cellular entry, and internalization and trafficking can be impacted by conjugation. Therefore, design of dendrimer−drug conjugates requires careful consideration to achieve successful drug delivery. KEYWORDS: nanomedicine, dendrimers, drug delivery, endocytosis, trafficking

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INTRODUCTION Dendrimers are nanoscale, highly branched polymers with unique physicochemical properties, including a high charge density, monodispersity, a predictable size, and a controllable number of surface groups making them ideal drug delivery vehicles.1 The surface groups can be conjugated to therapeutic compounds, imaging agents, and targeting moieties to create multifunctional nanomedicines tailored to have desired properties.2,3 Specifically, dendrimer−drug conjugates have the potential to increase drug solubility, enhance targeted drug accumulation, and limit off-target toxicities.4,5 Both poly(amidoamine) (PAMAM) and poly-L-lysine (PLL) dendrimers have been used to enhance the delivery of drugs with poor solubility and/or permeability.6−10 PAMAM dendrimers have been shown to permeate the intestinal epithelium and, therefore, may improve the oral bioavailability of Biopharmaceutics Classification System Class II and III compounds.11−13 To fully utilize the potential of dendrimers as delivery vehicles, particularly for oral drug delivery, the underlying transport and trafficking properties must be investigated. Dendrimer internalization and trafficking mechanisms have been extensively studied. We have shown that cationic and anionic PAMAM dendrimer uptake and transport are hindered in the presence of endocytosis inhibitors.14−16 PAMAM dendrimers also colocalize with markers for clatherin, early endosomes, and lysosomes.15,17 These studies all suggest that PAMAM dendrimers are internalized by both clatherin- and © 2015 American Chemical Society

caveolin-mediated endocytosis. In all of these studies, a fluorophore was conjugated to the dendrimer surface for detection. Because many PAMAM dendrimer properties are imparted by charge density, attaching fluorophores to the surface can interfere with the desired properties. The resultant charge shielding of the fluorophore may alter dendrimer uptake and trafficking, which would have eventual implications on the site of drug release. While PLL dendrimers have been used for several in vivo studies, a mechanistic analysis of cellular uptake and trafficking has yet to be conducted.6,7,18−20 PLL dendrimers provide a biodegradable alternative to PAMAM dendrimers while still maintaining similar physicochemical properties and are attractive drug delivery vehicles.18 In this study, we investigated the internalization and trafficking mechanisms of PLL dendrimers by measuring the impact of endocytosis inhibitors on dendrimer−cell interactions and visualizing intracellular trafficking. The dendrimers synthesized provided a unique opportunity for fluorophore conjugation not only to the surface but also to the dendrimer core. Therefore, we also investigated the impact of the fluorophore conjugation site on dendrimer properties. We compared the cytotoxicity, transport, uptake Received: Revised: Accepted: Published: 1961

November 17, 2014 March 26, 2015 May 6, 2015 May 7, 2015 DOI: 10.1021/mp500765e Mol. Pharmaceutics 2015, 12, 1961−1969

Article

Molecular Pharmaceutics

chromatography (SEC) with a 30:70 (v/v) acetonitrile/Tris buffer (pH 8) mobile phase was used to determine dendrimer polydispersity and confirm the absence of free fluorophore and other impurities. The OG content was determine by a fluorescence standard curve (λex = 490 nm; λem = 533 nm) measured using a Gemini XPS fluorescence microplate reader (Molecular Devices). The hydrodynamic diameter and ζ potential of each dendrimer were determined using a Malvern (Westborough, MA) Zetasizer Nano ZS. Samples were prepared in Hank’s Balanced Salt Solution (HBSS) at a concentration of 5 mg/mL, and measurements were taken in triplicate at 25 °C using an estimated viscosity of 0.8792 cP and a refractive index of 1.3316. Cell Culture. Caco-2 cells, a human colorectal adenocarcinoma cell line, were obtained from the American Type Culture Collection (Manassas, VA). Cells (passages 20−40) were grown at 37 °C with 5% CO2 and 95% relative humidity and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 units/mL penicillin, and 10000 μg/mL streptomycin. Medium was exchanged every other day, and cells were passaged using 0.25% trypsin/ethylene diamine tetraacetic acid at approximately 80−90% confluency. Dendrimer Cytotoxicity. Dendrimer cytotoxicity was assessed utilizing the WST-1 cell viability assay. Cells were seeded at a density of 50000 cells/well in clear 96-well plates and incubated under the conditions described above for 48 h until use. Dendrimer solutions (0.5, 1, and 1.5 mM) were prepared from 50 mM DMSO stocks and serially diluted in HBSS buffered with 10 mM 4-(2-hydroxyethyl)piperazine-1ethanesulfonic acid (HEPES). Cells were washed with HBSS and incubated with 100 μL of a dendrimer solution for 2 h. HBSS was used as a negative control to represent 100% cell viability, and 0.1% (v/v) Triton X-100 served as a positive control. After being treated, cells were washed with HBSS, and 100 μL of a 1:10 WST-1/HBSS mixture was added to each well. Cells were incubated for an additional 4 h, after which the absorbance was measured at 440 and 600 nm (background signal) with a SpectraMax Plus 384 absorbance microplate reader (Molecular Devices, Sunnyvale, CA). Results are reported as the percent viability compared to the HBSS control ± the standard deviation (SD). Statistical significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s test for multiple comparisons. Cellular Internalization. Uptake of dendrimers was measured in undifferentiated Caco-2 cells. Cells were seeded as previously described for cytotoxicity experiments. Dendrimer solutions (25 μM) were added to cells and incubated for 1 h at 37 °C. For inhibitor studies, cells were preincubated with 300 μM monodansyl cadaverine (MDC), 1 μM phenylarsine oxide (PAO), 4 μM filipin (FIL), 50 μM genistein (GEN), 10 mM methyl-β-cyclodextrin (MβCD), 50 μM dynasore (DYN), or 0.1 μM wortmannin (WORT) for 1 h followed by coincubation with inhibitor and dendrimer for an additional 1 h. After being treated, cells were washed twice with HBSS, and OG fluorescence was measured. Cells were then lysed with 70% (v/v) DMSO in buffered HBSS. A Bradford assay was used to determine the protein content for each well. Standard curves were prepared for each treatment condition to determine dendrimer content while eliminating any differences in fluorescence from dendrimer−inhibitor interactions and to account for the differences in fluorescent labeling. All inhibitors were confirmed to have >85% cell viability at the concen-

mechanisms, and trafficking of PLL dendrimers with Oregon Green (OG) 514 conjugated to either the dendrimer surface or the core. These studies will provide the knowledge necessary to rationally design dendrimer drug delivery systems while retaining the desirable properties that make dendrimers unique from traditional polymers, specifically with regard to oral drug delivery.

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EXPERIMENTAL SECTION Materials. Boc-Lys-ONp was obtained from ChemImpex (Wood Dale, IL). All cell culture supplies, OG514 carboxylic acid succinimidyl ester, transferrin-AlexaFluor 488 (AF488), lysosome-associated membrane protein 1 (LAMP1) antibody, secondary antibodies, and Prolong Gold with 4′,6-diamino-2phenylindole (DAPI) were purchased from Life Technologies (Grand Island, NY). Water-soluble tetrazolium salt 1 (WST-1) reagent was purchased from Roche (Indianapolis, IN). Early endosome antigen 1 (EEA1) antibody, endocytosis inhibitors, and all other chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Synthesis of OG Dendrimer Conjugates. PLL dendrimers were synthesized as previously described.18 Briefly, Boc-protected lysine functionalized with p-nitrophenol was reacted with L-lysine methyl ester in anhydrous dimethyl sulfoxide (DMSO) with distilled triethylamine (TEA) for 3 h at 75 °C. Once the reaction was complete, the product was extracted in dichloromethane (DCM) by being washed three times with ammonium chloride, three times with sodium bisulfate, and twice with brine. The Boc-protected product was then dried over magnesium sulfate, and the DCM was removed by rotary evaporation. The dendrimer was deprotected in a 1:1 DCM/trifluoroacetic acid (TFA) mixture for 1 h, after which the solvent was evaporated to obtain the deprotected firstgeneration dendrimer (G1). These steps were repeated with increasing reaction times using dry dimethylformamide (DMF) as the aprotic solvent to obtain G2 and G3. For G3, precipitation in 0.1 N sodium bicarbonate was used in the place of solvent extraction. The product was filtered, rinsed with acetonitrile, and dried under vacuum. For G3-OG dendrimers (OG attached to the dendrimer periphery), the dendrimer was deprotected as described above. After residual TFA had been thoroughly removed by evaporation, G3 and OG514 carboxylic acid succinimidyl ester were combined in a 1:1 ratio in DMF with TEA and reacted at room temperature overnight. Once the reaction was complete, solvent was removed by rotary evaporation. For G3EDA-OG dendrimers [OG attached to the dendrimer core via an ethylenediamine (EDA) linker], EDA was first added to the methyl ester at the dendrimer core. EDA was added to BOCprotected G3 in excess in DMF and reacted at room temperature overnight. The solvent was then evaporated, and the modified dendrimer was precipitated in acetonitrile and dried under vacuum. The product was reacted with a slight excess of OG514 carboxylic acid succinimidyl ester at room temperature overnight. After conjugation, the dendrimer was deprotected in a 4:1 DCM/TFA mixture for 1 h. Solvent was removed by rotary evaporation. Both dendrimers were dissolved in water and placed in dialysis tubing with a 0.5−1 kDa molecular mass cutoff (Spectrum Laboratories, Rancho Dominguez, CA) and dialyzed against water for 48 h. After dialysis, samples were lyophilized and stored at −20 °C. MALDI-TOF (Bruker Autoflex, Billerica, MA) was used to confirm all intermediates and products. Size exclusion 1962

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Molecular Pharmaceutics

Figure 1. Structures of (A) G3-OG and (B) G3-EDA-OG with the fluorophore and linker emphasized in bold.

trations used for experiments. Uptake results for native dendrimers are reported as the amount of dendrimer per the amount of protein in each well ± SD. An unpaired Student’s t test was used to confirm a statistically significant difference in uptake. Results of the inhibitors studied are presented as the percent uptake compared to the dendrimer alone ± SD. Statistically significant decreases between dendrimer uptake and uptake with inhibitor treatments were calculated by one-way ANOVA with Dunnett’s post-test. Differences between G3-OG and G3-EDA-OG were also compared using two-way ANOVA with Bonferroni correction. Transepithelial Transport. Transepithelial transport of the dendrimers was investigated in differentiated Caco-2 monolayers. Cells were seeded at a density of 80000 cells/well in 12-

well polycarbonate Transwell inserts (Corning, Tewksbury, MA) with a mean pore size of 0.4 μm and incubated for 21−28 days before being used. Monolayers were confirmed by measuring transepithelial electrical resistance (TEER) with a voltohmmeter (World Precision Instruments, Sarasota, FL), and only monolayers with a TEER of >500 Ω cm2 were used for experiments. Dendrimer transport was measured from the apical to basolateral compartment after a 1 h treatment with 25 μM G3-OG or G3-EDA-OG at 37 and 4 °C. Lucifer yellow (LY, 200 μM) was used as a control to which dendrimer permeability was compared. After incubation, samples were taken from the apical and basolateral chambers for fluorescence measurement. Results are reported as the percent of apparent permeability compared to LY ± SD. Statistical significance was 1963

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Molecular Pharmaceutics Table 1. Characterization of PLL Dendrimer−OG Conjugates dendrimer

MWa

PDIb

moles of OG per dendrimer

diameterc (nm)

ζ potentiald (mV)

G3-OG G3-EDA-OG

2449 2477

1.04 1.06

0.97 ± 0.20 0.58 ± 0.06

3.124 ± 0.043 3.017 ± 0.302

25.4 ± 1.3 17.2 ± 1.7

a Calculated from structure and confirmed by MALDI-TOF. bPolydispersity index, calculated from SEC data. cDetermined by dynamic light scattering. dMeasured by laser Doppler microelectrophoresis.

confirm dendrimer products, and fluorescence spectroscopy was employed to determine the OG content of each dendrimer. G3-OG had 0.97 ± 0.20 molecules of OG per dendrimer, while G3-EDA-OG had 0.58 ± 0.06. The polydispersity of each dendrimer conjugate was determined by SEC to be 1.04 and 1.06 for G3-OG and G3-EDA-OG, respectively. Dynamic light scattering was used to measure the hydrodynamic diameter of both dendrimers, and their ζ potential was determined by laser Doppler microelectrophoresis. G3-OG had a size of 3.124 ± 0.043 nm and a ζ potential of 25.4 ± 1.3 mV, while G3-EDAOG had a size of 3.017 ± 0.302 nm and a ζ potential of 17.2 ± 1.7 mV. Cytotoxicity of Dendrimers. Caco-2 cell viabilities after a 2 h treatment with conjugated and native PLL G3 dendrimers were compared to determine the impact of conjugation as well as conjugation site on cytotoxicity (Figure 2). A range was

calculated by one-way ANOVA with Tukey’s multiplecomparison test. Intracellular Trafficking. Dendrimer colocalization with intracellular organelles was measured in differentiated Caco-2 cells. Transferrin-AF488 was used as a control ligand. Monolayers were prepared and maintained as described above. Cells were washed twice with buffered HBSS followed by incubation in the apical compartment with 1 μM dendrimer or 250 μg/mL transferrin-AF488 for 30 min at 4 °C. After attachment to the cell surface had been allowed, cells were washed twice with ice-cold HBSS and incubated in HBSS at 37 °C for 5, 15, or 30 min to allow for internalization. Cells were then fixed in cold methanol at −20 °C for 15 min. After being washed three times with Dulbecco’s phosphate-buffered saline (DPBS), cells were permeabilized and blocked with 0.2% (v/v) Triton X-100 and 3% (w/v) bovine serum albumin (BSA) in DPBS for 1 h at room temperature. Once they had been permeabilized, cells were incubated with primary antibodies in blocking solution (2 μg/mL rabbit anti-EEA1 or rabbit antiLAMP1, both with 1.5 μg/mL mouse anti-occludin) for 1 h at 37 °C. Cells were then washed three times with DPBS and incubated with secondary antibodies in blocking solution (1:400 Cy5 goat anti-rabbit IgG and AF568 goat anti-mouse IgG) for 1 h at room temperature. After being stained, monolayers were washed three times with DBPS, excised, and mounted on glass slides with Prolong Gold containing DAPI. The mounting medium was allowed to cure for 24 h at room temperature, after which the slides were sealed and stored at 4 °C until they were imaged. Images were acquired using a Nikon A1 laser scanning confocal microscope. DAPI, OG/AF488, AF568, and Cy5 were excited with 404, 488, 561, and 641 nm lasers, respectively, and 450/50, 525/50, and 595/50 filter blocks were used. Four zstacks were collected for each image with the following parameters: Plan apo VC 60× oil objective, 38.0 μm pinhole, 4.6 μs pixel dwell, 2× line average, 3× optical zoom, 1 μm zstep size, and 512 × 512 image size. Images were processed using Volocity 3D Image Analysis Software version 6.3 (PerkinElmer, Waltham, MA). Colocalization between OG/ AF488 and the nucleus, tight junctions, early endosomes, and lysosomes was calculated in the software by thresholding each channel between 10 and 100%. Mander’s colocalization coefficient (Mx) was used to represent the fraction of green voxels that colocalized with blue, red, or pseudopurple. Results are displayed as Mx ± SD.

Figure 2. Caco-2 cell viability after a 2 h incubation with native and conjugated PLL dendrimers. Values are means ± SD (n = 4). Asterisks denote a statistically significant decrease in cell viability compared to the untreated control (p < 0.001).

chosen to encompass known nontoxic and highly toxic native G3 concentrations. Conjugation of a fluorophore to a terminal amine resulted in decreased dendrimer toxicity. G3-OG was nontoxic up to 1 mM and showed less toxicity than G3 alone at 1.5 mM. Conjugating OG to the dendrimer core, however, resulted in an increase in dendrimer toxicity with the lowest concentration tested decreasing cell viability more than the 1 mM G3 treatment. Impact of the OG Conjugation Site on Dendrimer Uptake and Transport. Internalization and transepithelial transport of G3-OG and G3-EDA-OG were compared to establish the impact of dendrimer conjugation on these properties. Accounting for differences in fluorescent labeling, we found Caco-2 cells had nearly double the uptake of G3EDA-OG compared to that of G3-OG within 1 h of treatment (Figure 3). For transepithelial transport, G3-OG achieved

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RESULTS Synthesis and Characterization of G3-OG and G3EDA-OG. PLL G3 dendrimers with OG514 located either on the dendrimer periphery or at the dendrimer core were synthesized to determine the intracellular uptake and trafficking of PLL dendrimers and to ascertain the impact of fluorophore location on these mechanisms. The structures of both dendrimers are illustrated in Figure 1, while the physicochemical properties are listed in Table 1. MALDI-TOF was used to 1964

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Figure 3. Uptake of dendrimers in Caco-2 cells after a 1 h treatment. Results are means ± SD (n = 4).

significantly higher transport than G3-EDA-OG and the LY control in differentiated monolayers (Figure 4). G3-OG also

Figure 5. Percent uptake of G3-OG (white bars) and G3-EDA-OG (gray bars) in Caco-2 cells in the presence of endocytosis inhibitors. Results are means ± SD (n = 4). One asterisk, two asterisks, and three asterisks indicate a statistically significant decrease in uptake compared to dendrimer uptake alone (p < 0.05, p < 0.01, and p < 0.001, respectively).

therefore, PLL dendrimers likely utilize caveolae as a means of internalization. In addition to specific inhibitors of clatherin- or caveolinmediated endocytosis, we also studied compounds that inhibit both mechanisms as well as a macropinocytosis inhibitor. MβCD, a cyclic glucopyranoside oligomer that extracts cholesterol from the plasma membrane, was used to establish the impact of cholesterol in dendrimer uptake.31,32 Treatment with MβCD resulted in a significant decrease in dendrimer uptake regardless of fluorophore location. Cholesterol is important for internalization in both clatherin-coated pits and caveolae; therefore, dendrimers are likely internalized in part through both mechanisms.33,34 We also investigated the impact of DYN, a dynamin inhibitor.35 Dynamin2 is a GTPase responsible for vesicle fission in coat-dependent and -independent endocytic pathways.36,37 We again saw a significant decrease in dendrimer internalization, further supporting the importance of clatherin and caveolae for both dendrimers. WORT was chosen as an inhibitor of macropinocytosis, a nonselective endocytosis route commonly utilized by macromolecules.38 WORT prevents macropinocytosis by inhibiting phosphoinositide 3-kinase, a protein necessary for the closure of macropinosomes.39,40 Dendrimer uptake was diminished by WORT treatment, suggesting the role of nonspecific internalization of dendrimers in addition to the selective clatherin- and caveolin-mediated mechanisms. Intracellular Trafficking and Visualization of Dendrimers Interacting with Monolayers. Along with establishing mechanisms of PLL dendrimer internalization, we also investigated the intracellular trafficking of the dendrimers in polarized Caco-2 monolayers. Colocalization coefficients were calculated for the overlap of dendrimers or transferrin with the nucleus (DAPI), tight junctions (occludin), early endosomes (EEA1), and lysosomes (LAMP1) (Figure 6). G3-OG showed rapid uptake into the cell and localized early with the nucleus, endosomes, and lysosomes. As incubation time increased, little change in dendrimer localization was noted, suggesting that G3-OG internalization is not specific to mechanisms involved in endosomal and lysosomal trafficking. G3-EDA-OG displayed consistent uptake within the nucleus, with localization to the tight junctions and endosomes increasing with time. Colocalization with lysosomes was substantially higher than that with

Figure 4. Transport of dendrimers across differentiated Caco-2 monolayers at 37 °C (white bars) and 4 °C (gray bars). Results are means ± SD (n = 4). Asterisks denote statistical significance between different 37 °C treatments and between 37 and 4 °C samples of the same treatment (p < 0.001).

displayed a significant decrease in transport at 4 °C, while G3EDA-OG experienced only a minimal decrease. Both of these studies illustrated the impact the conjugation site can have on eventual dendrimer drug delivery. Dendrimer Internalization in the Presence of Endocytosis Inhibitors. Cellular uptake of G3-OG and G3-EDAOG was assessed utilizing a panel of inhibitors to investigate clatherin-, caveolin-, cholesterol-, and dynamin-mediated endocytosis as well as macropinocytosis (Figure 5). Standard curves for each dendrimer−inhibitor combination were used to accurately determine dendrimer uptake. MDC and PAO were used to inhibit clatherin-mediated endocytosis. MDC increases clatherin polymerization, creating stabilized pits that are unable to form vesicles.21−23 PAO is suspected to inhibit clatherincoated pit internalization as well by causing a depletion of diacylglycerol, a small signaling lipid that affects the curvature of membrane bilayers and eventual vesicle scission.24−26 MDC had no impact on uptake for either dendrimer. PAO inhibited uptake of G3-EDA-OG but not G3-OG, suggesting that dendrimer uptake may partially be mediated by clatherin and affected by fluorophore location. To study caveolin-mediated endocytosis, FIL and GEN were used. FIL forms a complex with cholesterol in noncoated membrane invaginations rich in cholesterol, such as those associated with caveolae, disrupting vesicle formation.27,28 GEN interferes with caveolin-mediated endocytosis by inhibiting tyrosine kinases required for actin cytoskeletal reorganization and dynamin II recruitment, essential events for internalization in caveolae.29,30 While FIL did not decrease G3-OG uptake, G3-EDA-OG uptake was reduced. GEN lowered the uptake of both dendrimers; 1965

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Figure 7. Interaction of dendrimers and transferrin with differentiated Caco-2 monolayers. Images are representative of the 30 min time point used to calculate colocalization: blue, nucleus; red, tight junction; purple, endosome−lysosome; and green, dendrimer−transferrin.

punctate regions resembling vesicles, supporting the inhibitor results that showed that dendrimers utilize vesicular endocytosis for internalization. Additionally, G3-EDA-OG showed localization with the cell nucleus, which is unsurprising given the use of cationic dendrimers as transfection agents.44

Figure 6. Intracellular trafficking of (A) G3-OG, (B) G3-EDA-OG, and (C) transferrin-AF488 in Caco-2 monolayers. Colocalization with the nucleus (DAPI), tight junctions (occludin), endosomes (EEA1), and lysosomes (LAMP1) is shown at 5 min (white), 15 min (gray), and 30 min (black). Results are means ± SD (n = 4).

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DISCUSSION PAMAM dendrimers have been shown to traverse intestinal epithelia while carrying poorly bioavailable drugs.11−13 While PAMAM dendrimer internalization and trafficking have been established,14−17 the mechanisms of PLL dendrimer uptake and trafficking have yet to be investigated. Localization of drug delivery systems can impact drug release, degradation, and toxicity;45 therefore, understanding the mechanisms of cellular entry and intracellular trafficking is exceedingly important. In this study, we established mechanisms of uptake utilized by PLL dendrimers as well as the intracellular localization of these dendrimers after internalization. We also compared the uptake and transport of dendrimers with a fluorophore conjugated to either the dendrimer surface or the core to establish the impact of conjugation on dendrimer properties. Studies of PAMAM dendrimer internalization rely on monitoring a fluorophore conjugated to the dendrimer surface instead of the dendrimer itself. Although fluorophore

other markers with a marginal decrease between 15 and 30 min. In contrast, transferrin had minimal nucleus and tight junction colocalization and was primarily present within the endosomes and lysosomes. Lysosomal localization remained consistent across the time points that were investigated, suggesting pathway saturation. While these results were different from previously published findings in undifferentiated cells, polarized monolayers have distinctly different protein localization at the apical and basolateral domains.15,41−43 Figure 7 demonstrates the interaction of dendrimers and transferrin with monolayers after a 30 min incubation. Green fluorescence was generally present in the upper half of the cells, and all images showed internalization. G3-EDA-OG and transferrin visually exhibited an interaction with the monolayer much stronger than that of G3-OG. All three treatments have 1966

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Molecular Pharmaceutics

caveolae and lipid rafts in PLL dendrimer uptake. Decreased internalization in the presence of WORT, an inhibitor of nonspecific macropinocytosis, highlighted the ability of dendrimers to enter cells through a variety of routes. Like PAMAM dendrimers, PLL dendrimers are not relegated to a single means of internalization, and endocytosis is a result of a combination of mechanisms. Intracellular trafficking studies were used to further elucidate the environments that may be encountered by the dendrimers after internalization. Knowledge of the pH and enzymes a drug delivery system will experience, which vary by intracellular compartment, is crucial for proper drug release.45 PAMAM dendrimers have been shown to colocalize with endosomal and lysosomal markers in Caco-2 cells;15,17 however, the trafficking of PLL dendrimers has yet to be investigated. Also, the previous studies were conducted in undifferentiated cells in which trafficking can occur in only one direction. Our studies were conducted in polarized monolayers that allowed for trafficking to and from both the apical and basolateral compartments. All treatments showed minimal colocalization with occludin, and G3-OG and transferrin had little colocalization with the nucleus. G3-EDA-OG had consistent nuclear localization within 5 min of treatment. Cationic dendrimers are proven gene transfection agents;44 therefore, localization of the fully charged dendrimer to the nucleus was not unexpected. The patterns of colocalization with early endosomes and lysosomes were unique to each treatment. G3-OG had rapid endosomal trafficking followed by a decline before increasing again, while lysosomal trafficking showed the reverse pattern. For G3-EDAOG, localization in the endosomal compartment slowly increased with time while lysosomal compartment localization was rapid with a large portion of dendrimer present. In polarized cells, EEA1 has been shown to associate with basolateral endosomes that do not associate with apical endosomes.42 From our transport study, G3-OG was capable of traversing the epithelial monolayers while G3-EDA-OG had much lower transport. Therefore, G3-OG may be trafficked to the basolateral endosomes faster than G3-EDA-OG, which takes much longer to reach the basolateral side of the cell because of electrostatic interactions. Localization with EEA1 decreased as G3-OG was efficiently exocytosed until more dendrimer could reach the basolateral domain. G3-EDA-OG exhibited a higher level of lysosomal accumulation than G3OG, which could have implications on drug delivery vehicle design. The control ligand transferrin did not colocalize with endosomes until 15 min while being consistently present in the lysosomes. Transferrin is typically internalized via clatherinmediated endocytosis from the basolateral membrane;48 therefore, the delay in endosomal localization may have been a result of the apical treatment. Different subcellular compartments possess different pHs and enzymes. Drugs are commonly conjugated to dendrimers via either pH-sensitive or enzyme-cleavable linkers;49 therefore, knowledge of subcellular trafficking is essential for the selection of the correct conjugation strategy for optimal drug release. Our results indicate that PLL dendrimers exploit a variety of internalization mechanisms, preferentially utilizing cholesterolmediated uptake and macropinocytosis. We also demonstrated the impact of conjugation site on uptake, transport, and subcellular localization. These studies highlight the importance of rationally designing dendrimer delivery systems to achieve successful drug delivery. The conjugation site can be chosen on the basis of the desired delivery location, and drug linkers can

conjugation likely influences the uptake, transport, and routing of the dendrimer, conjugation of the drug would likely induce the same effects; therefore, fluorophores such as fluorescein and other fluorinated analogues may provide good substitutes for studying the impact of small hydrophobic drugs on dendrimer delivery properties. Our PLL dendrimers contained an unbranched methyl ester group at the core providing an additional option for conjugation compared to traditional surface conjugation strategies; therefore, we conjugated OG to either the dendrimer periphery or the dendrimer core via an EDA linker. Comparing the cytotoxicity of the conjugated dendrimers to native PLL G3, we found that the conjugation site had opposite consequences on dendrimer toxicity. G3-OG was less toxic to the cells than the native dendrimer. Other studies have reported similar findings in which conjugation to the dendrimer surface resulted in a decreased level of cell death.46 Even with one fluorescein molecule per dendrimer, the cytotoxicity of PAMAM G4 was decreased at high concentrations, and increasing the ratio of fluorophore to dendrimer further ameliorated cytotoxicity.46 Considering the decreased ζ potential of G3-EDA-OG with no apparent changes in the hydrodynamic diameter, conjugating OG to the dendrimer core may have created amphiphilic dendrimers capable of dimerizing to form a hydrophobic pocket around the fluorophores. As a dimer, the core-conjugated dendrimer would present double the amount of surface amines that may cause increased toxicity; however, further characterization would be needed for confirmation. Although G3-EDA-OG has a higher cytotoxicity, the concentrations tested are much higher than concentrations that would be needed for drug delivery in vivo.13 In addition to cytotoxicity, we also investigated the impact of the fluorophore site on PLL G3 uptake and transport. Surface conjugation significantly reduced the uptake of PLL G3 compared to core conjugation. G3-EDA-OG experienced more than double the amount of uptake compared to G3OG. For transepithelial transport, the opposite effect was seen. G3-OG had an apparent permeability >3 times that of G3EDA-OG and the LY control. We also investigated transport at 4 °C and found that G3-OG transport was significantly decreased, indicating that energy-dependent mechanisms are in part responsible for dendrimer transport. Only a marginal difference was seen for G3-EDA-OG at 4 °C either because transport was minimal to begin with or because G3-EDA-OG cellular interactions were primarily electrostatic. Cationic dendrimers have been shown to predominantly interact with membranes electrostatically,47 and dimerization of G3-EDAOG would change the shape and surface charge of the dendrimer, potentially allowing for a greater electrostatic interaction with the cell surface while G3-OG is transported through the cell. To further investigate the impact of fluorophore conjugation and establish internalization mechanisms of PLL dendrimers, we investigated dendrimer uptake in the presence of a panel of inhibitors of different endocytosis mechanisms. Dendrimer endocytosis occurred equally between specific and nonspecific mechanisms. Inhibitors of clatherin-mediated endocytosis had the least impact on uptake, while caveolin-mediated endocytosis inhibitors caused more substantial decreases. In general, inhibitors for both mechanisms were more potent for G3-EDAOG; however, differences between G3-OG and G3-EDA-OG were not statistically significant. Inhibitors of both cholesteroland dynamin-mediated endocytosis significantly reduced dendrimer internalization, further implicating the role of 1967

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Article

Molecular Pharmaceutics

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be designed to be compatible with the most likely dendrimer accumulation site.

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CONCLUSION In this work, we reported the detailed mechanisms of endocytosis and intracellular trafficking of PLL dendrimers in Caco-2 cells and the impact of fluorophore conjugation site on these mechanisms. We found that PLL dendrimers primarily employed caveolin-, cholesterol-, and dynamin-mediated endocytosis while also being taken up through nonspecific macropinocytosis. Dendrimers were localized in both endosomes and lysosomes, with core-conjugated dendrimers exhibiting higher localization in these vesicles. Knowledge of these mechanisms will be beneficial in achieving successful drug release and therapeutic results. Additionally, the drug conjugation site may be tailored to the desired delivery route. In summary, the results of this work will aid in the rational design of PLL dendrimers as drug delivery carriers.

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AUTHOR INFORMATION

Corresponding Author

*Department of Pharmaceutical Sciences, University of Maryland, Baltimore, 20 Penn St., Health Sciences Facility II, Room 543, Baltimore, MD 21201. E-mail: [email protected]. Telephone: (410) 706-0103. Fax: (410) 706-5017. Notes

The authors declare no competing financial interest.

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DOI: 10.1021/mp500765e Mol. Pharmaceutics 2015, 12, 1961−1969